We want to understand how eukaryotic cells direct specific proteins along a pathway leading to secretion of those proteins. Current hypotheses suggest that the amino acid sequences of proteins destined for secretion trigger the sequestration of these proteins inside endoplasmic reticulum. We shall examine the sequestration of one typical secreted protein, using cloned DNA that codes for the particular protein. We are developing several cellular and cell-free systems that will allow us to transcribe the cloned DNA into RNA, translate the RNA into protein, and then sequester the protein inside endoplasmic reticulum. SV40 DNA will be coupled to the cloned DNA to introduce the cloned DNA into mammalian cells in tissue culture; plasmids will be used to introduce the cloned DNA into bacteria. Further, in a cell-free system cloned DNA will be transcribed and translated, and then the protein so produced will be sequestered inside dog pancreas microsomal membranes. Using recombinant DNA technology, we shall then modify the cloned DNA sequences systematically, and test the functional significance of these changes in our cellular and cell-free assay systems. By noting which modifications of DNA result in characteristic abnormalities of sequestration, we hope to determine how secreted proteins direct their own sequestration. Viewed from the more general perspective of cell biology, we want to investigate one way that cells organize their constituents into discrete compartments.